CN113447659B - Kit for detecting anti-proteasome subunit alpha 1-IgG antibody - Google Patents
Kit for detecting anti-proteasome subunit alpha 1-IgG antibody Download PDFInfo
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- CN113447659B CN113447659B CN202110743551.7A CN202110743551A CN113447659B CN 113447659 B CN113447659 B CN 113447659B CN 202110743551 A CN202110743551 A CN 202110743551A CN 113447659 B CN113447659 B CN 113447659B
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- subunit alpha
- proteasome subunit
- type1
- antigen
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Abstract
The invention provides a kit for detecting an anti-proteasome subunit alpha 1-IgG antibody, which consists of an antigen protein Protezomesubunit alpha type 1(proteasome subunit alpha 1), a solid phase carrier, a labeled antibody, an antigen and antibody diluent, a sample dilution buffer solution, a substrate color developing agent, a washing solution, a standard substance and positive and negative quality control substances. The kit provided by the invention detects the anti-proteasome subunit alpha 1-IgG antibody in the serum to be detected by using an indirect method reaction principle and combining with magnetic particle chemiluminescence immunoassay. The invention identifies the autoantibody aiming at the target antigen proteasome subunit alpha 1 in the serum of the patient with autoimmune nephrotic syndrome for the first time. The invention provides a basis for the molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to proteasome subunit alpha 1 and proteasome subunit alpha 1-IgG autoantibody at home and abroad.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a kit for detecting an anti-proteasome subunit alpha 1-IgG antibody.
Background
In recent years, the types of kidney diseases of children are more and more, wherein the incidence rate of autoimmune nephrotic syndrome is the highest, and the physical and mental health of children is seriously harmed. Autoimmune nephrotic syndrome is a clinical syndrome which is characterized by a large amount of proteinuria, hypoproteinemia, high edema and the like caused by the fact that the filtration of plasma proteins is increased due to the increase of the permeability of a glomerular filtration membrane, and a large amount of proteinuria is caused. The Ali et al observed that after transplantation of the kidney from the refractory morbid nephrotic syndrome patient, the subject had a normal kidney function without any proteinuria, and thus it was shown that the cause of the morbid nephrotic syndrome was not all in the kidney itself, but probably mainly in the internal environment of the patient. In addition, except for children who suffer from partial gene defects, the conditions of most of the children suffering from autoimmune nephrotic syndrome are improved after the treatment of hormones and immunosuppressants, which indirectly proves that the disease is closely related to the autoimmunity of patients.
In recent years, it has been found that B cell dysfunction also plays an important role in autoimmune nephrotic syndrome. In recent years, a plurality of multi-center clinical research results in the world show that Rituximab (Rituximab, RTX) can be successfully used for treating minimal-lesion nephrotic syndrome, and particularly, the Rituximab (RTX) has a good treatment effect in the aspect of treating refractory nephrotic syndrome. However, it has been found that in the course of treatment of hormone dependent nephrotic syndrome with rituximab, its B cell scavenging effect is maintained for about 5 months, and the disease will recur in patients with B cell numbers rising again between 6 and 7 months. Therefore, pathological B cell clones exist in the body of the patient with the autoimmune nephrotic syndrome, and are identified and accurately eliminated, so that the recovery of the autoimmune nephrotic syndrome is facilitated, and the risk of humoral immune deficiency of the patient caused by indiscriminate B cell elimination by means of rituximab and the like is reduced. However, to date, the target antigens targeted by pathological B cells in children with autoimmune nephrotic syndrome are still not well understood. Pathologically, minimal morbid nephropathy or focal segmental glomerulosclerosis is considered podocytosis resulting in massive proteinuria due to loss or alteration of podocyte function. Podocytes, the renal glomerular epithelial cells, which adhere to the outside of the glomerular basement membrane, are the last barrier to prevent protein loss, and podocyte injury often causes massive proteinuria.
Proteasomes (proteosomes) are macromolecular complexes that degrade proteins within cells, the primary function of which is to degrade proteins that are not needed or damaged by the cell. Proteasome subunit alpha-1 (Proteasome subunit alpha type 1) is a member of the Proteasome family, and the Proteasome subunit alpha type1 protein is currently studied in cancer. If the research shows that the protein of protease subenit alpha type1 in the Proteasome pathway is closely related to the control of cell cycle progression and apoptosis, the expression of the protein subenit alpha type1 is obviously reduced in hepatocellular carcinoma tumor tissue, and the protein subenit alpha type1 can be a potential biomarker of hepatocellular carcinoma (Jian Qin1, et al. identification of the protein subenit alpha type-1as a non-biological marker in-associated hepatocellular carcinoma. mu. of hepatocellular carcinoma and HBV 7812. 78120.). Yang Qian et al report that the protein subnitrile alpha type1 protein may be a potential biomarker for colon cancer (Yang Qian, Roehrl Michael H, Wang J, et al. genomic profiling of anti-inflammatory-inducing antibodies in tumor tissue antigens protein subnitrile alpha type1, LAP3, ANXA3, and maps as a color marker markers. journal [ J ] Oncotarget Volume 9, Issue 3.2018.PP 3996-4019.).
However, there is no report on the expression of proteosome subustit alpha type1 and the presence of autoantibodies to proteosome subustit alpha type1 in nephrotic syndrome. In addition, in the prior art, the application of the protein subunit subbnit alpha type1 or the autoantibody thereof as a serological marker in autoimmune nephrotic syndrome is not involved. A study for identifying autoimmune immune nephrotic syndrome by detecting serum anti-proteosome subbunit alpha type1-IgG antibodies was blank.
Disclosure of Invention
The invention aims to provide a kit for detecting an anti-Proteasome subunit alpha 1-IgG antibody, which is a detection kit based on a target point protease subunit subbunit alpha type1 and a corresponding autoantibody thereof. The kit can detect the autoantibody from tissues (kidney biopsy tissues) or body fluids (particularly blood, plasma and serum) through immunoreaction with an antigen protein Proteomer subbunit alpha type1 (particularly shown according to a sequence identification number SEQ ID NO. 1).
The kit comprises an antigen protein Proteonome subbunit alpha type1, a solid phase carrier, a labeled antibody such as an enzyme label, a chemiluminescent reagent label or a biotin labeled secondary antibody, an antigen diluent, a sample diluent buffer, an antibody diluent, a substrate color developing agent, a washing solution, a standard substance, a positive quality control substance and a negative quality control substance.
The sequence of the antigen protein proteaseome subunit alpha type1 is shown in SEQ ID NO. 1: MFRNQYDNDVTVWSPQGRIHQIEYAMEAVKQGSATVGLKSKTHAVLVALKRAQSELAAHQKKILHVDNHIGISIAGLTADARLLCNFMRQECLDSRFVFDRPLPVSRLVSLIGSKTQIPTQRYGRRPYGVGLLIAGYDDMGPHIFQTCPSANYFDCRAMSIGARSQSARTYLERHMSEFMECNLNELVKHGLRALRETLPAEQDLTTKNVSIGIVGKDLEFTIYDDDDVSPFLEGLEERPQRKAQPAQPADEPAEKADEPMEH are provided.
The proteosome subbunit alpha type1 antigenic protein of the invention may be a fusion protein, using a tag with certain biological or physical functions, in particular the N-terminus or the C-terminus. The existence of the tags is beneficial to the purification, fixation and precipitation of antigen protein. In a preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, e.g., the tag peptide is selected from the group consisting of: a GST tag, a c-Myc tag, a His tag, a Flag tag, or a biotin tag.
According to the invention, the antigen protein proteosome subbnit alpha type1 is fixed on the solid phase carrier, and the proteosome subbnit alpha type1 antigen protein is fixed on the solid phase carrier by utilizing physical adsorption or covalent bond combination or chemical combination. Preferred solid supports include: nitrocellulose membrane, magnetic beads and enzyme-labeled microporous plate.
In one embodiment of the invention, the standard substance and the positive quality control substance are recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-protease subunit alpha type1-IgG antibody extracted from patient serum is used as the positive quality control substance and the standard substance, and the serum of a healthy examinee is the negative quality control substance.
According to the invention, the antigen protein proteosome subbunit alpha type1 can be expressed in bacteria such as Escherichia coli, yeast and mammalian cells.
According to the invention, the antigen protein proteosome subbunit alpha type1 is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography and hydrophobic column.
According to the invention, the biological sample is a sample comprising autoantibodies, selected from the group consisting of whole blood, serum, plasma, urine, lymph fluid, pleural effusion and ascites. Preferably mammalian (human) serum.
The detection kit further comprises a substrate color developing agent, an antigen diluent, a sample dilution buffer solution, an antibody diluent and a washing solution. The substrate color developing agent is TMB, AMPPD, 4-MUP and BCIP; the antigen diluent is 1 XPBS pH7.4 containing 163mM NaCL and 1% TritonX-100; the sample dilution buffer was 0.01M PBS ph7.4 containing 10% BSA; the antibody diluent is 0.01M PBS pH7.4 containing 1M D-glucose, 2% glycerol, 0.35% Tween 20; the washing solution is as follows: 1 XPBS pH7.4 containing 163mM NaCL, 10% glycerol, 1% TritonX-100.
In a preferred embodiment, "immobilized" as described herein refers to binding to a solid support that is insoluble in water of the proteosome subunit type1 antigenic protein, the solid support or support being insoluble in water, more preferably by covalent bonding, electrostatic interaction, hydrophobic interaction, or by disulfide bond interaction, most preferably by one or more covalent bonds. The immobilization may be by direct immobilization, e.g. by filtration, centrifugation or chromatography, and the immobilized molecules are separated from the aqueous solution together with the insoluble support. Also includes fixing the Proteasome subbunit alpha type1 antigen protein in a reversible or irreversible mode. For example, the antigenic protein is immobilized to the carrier by a cleavable covalent bond (e.g., a disulfide bond that can be cleaved by addition of a thiol-containing reagent), which is reversible. In addition, if the antigenic protein is immobilized to the support by a covalent bond that does not cleave in aqueous solution (bond formed by reaction of epoxide group with amine group coupling lysine side chain to affinity column), the immobilization is irreversible. Fixation may also be indirect: such as fixing an antibody having a specific affinity for the antigen protein, and then forming an antigen protein-antibody complex for the purpose of fixing.
The method for fixing the antigen protein proteosome subbnit alpha type1 is a direct coating method: (1) the antigen protein proteosome subbnit alpha type1 is combined on a nitrocellulose membrane or a polystyrene micropore plate in a physical adsorption mode or a non-covalent bond; (2) the magnetic particle with the carboxyl functional group is combined with the amino group of the antigen protein Proteasome tributary alpha type1, and the antigen protein Proteasome tributary alpha type1 is combined on the magnetic particle in a chemical coupling mode.
The labeled antibody selected by the invention can be Horseradish Peroxidase (HRP) labeled anti-human IgG antibody, acridinium ester labeled anti-human IgG antibody and biotin labeled anti-human IgG antibody.
The invention adopts a gene recombination prokaryotic expression method to successfully express and purify a recombinant protein Protezome subunit alpha type1, and develops a kit suitable for detecting serum anti-Protezome subunit alpha type1-IgG antibodies of patients with autoimmune nephrotic syndrome by taking the recombinant protein Protezome subunit alpha type 1as an antigen protein in the kit. Comprises a detection kit for qualitatively or quantitatively analyzing and detecting an anti-proteosome subbnit alpha type1-IgG antibody in human serum.
A principle of a kit for detecting an anti-Proteomer subunit alpha type1-IgG antibody in serum utilizes an indirect method reaction principle, firstly, a Proteomer subunit alpha type1 antigen is adsorbed to a solid phase carrier to serve as a coating antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled secondary antibody is added for reaction, if anti-Proteomer subunit alpha type1-IgG antibody is contained in the serum to be detected, a coating antigen Proteomer subunit alpha type 1-anti-Proteomer subunit alpha type1-IgG antibody-labeled anti-human IgG antibody ternary complex of the serum to be detected is formed, and finally, an optical signal is detected by utilizing a photochromogenic method, a chemiluminescence method and a fluorescence method, so that the aim of qualitatively or quantitatively analyzing the anti-Proteomer subunit alpha type1-IgG antibody in the human serum is achieved.
The kit provided by the invention is used for detecting an anti-proteosome subunit alpha type1-IgG autoantibody in part of autoimmune nephrotic syndrome patients for the first time, and the target antigen aimed by the autoantibody is determined to be Proteasome subunit alpha-1 (proteosome subunit alpha type 1) on podocytes. Therefore, the kit can be used for detecting the anti-protesome subbunit alpha type1-IgG autoantibody, and provides a basis for researching the autoimmune nephrotic syndrome.
Compared with the prior art, the kit has the advantages that:
(1) at present, the related protease subnitrile alpha type1 and anti-protease subnitrile alpha type1-IgG antibodies of kidney disease patients at home and abroad are only limited to molecular mechanism research, and the level of the antibodies in the serum of the patients is not quantitatively detected. The invention identifies the autoantibody aiming at the proteosome subbnit alpha type1 for the first time, and invents a detection kit aiming at the anti-proteosome subbnit alpha type1-IgG autoantibody, thereby filling the blank at home and abroad.
(2) The kit relates to qualitative analysis of an anti-proteosome subbunit alpha type1-IgG antibody in human serum, wherein the solid-phase membrane immunoassay is simple to operate, the reagent dosage is less, and the kit is saved by about 10 times compared with the traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely close to 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane; the NC membrane with adsorbed antigen or antibody or existing result can be preserved for a long time (half a year at-20 ℃), and the activity of the NC membrane is not influenced; in addition, the kit for qualitatively detecting the anti-proteosome subbnit alpha type1-IgG antibody in the human serum by the solid-phase membrane immunoassay is introduced into a biotin-avidin amplification system, so that the detection sensitivity is greatly improved.
(3) The kit for quantitatively detecting the anti-proteosome subbunit alpha type1-IgG antibody in human serum, which is related by the invention, utilizes magnetic particles as solid phase carriers, the diameter of the magnetic particles is only 1.0 mu m, so that the coating surface area is greatly increased, the adsorption quantity of antigens is increased, the reaction speed is improved, and the cleaning and separation are simpler and more convenient, thereby reducing pollution and reducing the probability of cross infection. On the other hand, the acridine ester luminescent agent is adopted to directly mark the anti-human IgG, the chemical reaction is simple and quick, and no catalyst is needed; the acridinium ester chemiluminescence is of the scintillation type by initiating the luminescent reagent (H)2O2NaOH) can reach the maximum after 0.4s, the half-life period is 0.9s, the process is basically finished in 2s, and the rapid detection is convenientAnd (6) measuring.
Drawings
FIG. 1: the Proteasome subustit alpha type1 protein on podocyte is the target antigen for the autoantibody in autoimmune nephrotic syndrome patients. FIG. 1A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; FIG. 1B: the first antibody is a two-dimensional electrophoresis protein spot of serum of an autoimmune nephrotic syndrome patient; FIG. 1C: mass spectrometry identification of target antigen Proteasome subunit alpha type1 protein.
FIG. 2: SDS-PAGE identification picture of expressed recombinant protein Proteasome subunit alpha type 1.
FIG. 3: the solid-phase membrane immunoassay kit is used for detecting an anti-proteosome sunburnt alpha type1-IgG antibody in serum of an autoimmune nephrotic syndrome patient.
FIG. 4 is a drawing: schematic diagram of the principle of detecting anti-protesome subbunit alpha type1-IgG antibody by using magnetic particle chemiluminescence immunoassay kit.
FIG. 5: schematic diagram of antigen protein proteome subbunit alpha type1 coated carboxyl magnetic particles.
FIG. 6: detection of anti-proteosome subbunit alpha type1-IgG antibodies in patients with various renal diseases, wherein NS: autoimmune nephrotic syndrome, HSP: allergic purpura, HSPN: purpuric nephritis, IgAN: IgA nephropathy, NC: a healthy child.
FIG. 7: the application value of the antibody of the anti-proteosome subbnit alpha type 1as a serological marker for diagnosing autoimmune nephrotic syndrome is evaluated by an ROC curve.
Detailed Description
The invention is further described below with reference to the following figures and specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1Proteasome subunit alpha type1 protein on podocytes is a target antigen for autoantibodies to target in autoimmune nephrotic syndrome patients
According to the invention, a large number of clinical and molecular mechanism researches at the early stage are carried out, the serum IgG level of a patient with nephrotic syndrome is found to be high for the first time, and the proteosome subbunit alpha type1 on podocytes is proved to be a target antigen for the autoantibody in the patient with autoimmune nephrotic syndrome. Therefore, the detection of the presence and quantitative level of anti-proteosome subbunit alpha type1-IgG antibodies in serum is beneficial to the early identification of autoimmune nephrotic syndrome, especially to the screening of patients with relevant symptoms. Specifically, the following (1) extraction of total protein of glomerular podocyte is carried out: the podocyte strain (MPC5) was cultured, washed 2-3 times with PBS, then sufficiently lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (# ab 65621; Abcam, 1: 200 dilution), and then the samples were centrifuged at 12000g for 30min at 4 ℃. And collecting supernatant, namely collecting the total protein of the glomerular podocyte. The total protein concentration of the collected glomerular podocytes was determined using the BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: extracting total protein of glomerular podocyte, performing two-dimensional electrophoresis, transferring to nitrocellulose membrane, incubating with serum of healthy people and autoimmune nephrotic syndrome patients as primary antibody, and developing with secondary antibody as shown in fig. 1A and 1B. (3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after visualization in step (2), protein spots which were strongly positive for nephrotic syndrome patients on two-dimensional electrophoresis gel and negative or weakly positive for healthy persons were selected, the selected protein spots were excised from the gel, the dried gel was digested with trypsin (0.1. mu.g/. mu.l), 10. mu.l of 25mM ammonium bicarbonate was added to the reaction mixture, incubated overnight at 37 ℃, and peptides were then extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptides were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass spectrum, which was identified as the protease subunit alpha type1 protein, as shown in FIG. 1C.
Example 2 expression and purification of recombinant antigenic protein Proteonome subunit alpha type1
The gene of the protein coding the protease subnitrile alpha type1 is used as a template by utilizing a genetic engineering method, PCR amplification is carried out, and then an expression vector is constructed for protein expression. The expressed antigen protein of the invention contains a tag peptide of GST tag. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve and the like, and finally the molecular weight of the recombinant protein Proteasome subunit alpha type1 is identified by SDS-PAGE, and is shown in figure 2, wherein the molecular weight is 56 KDa.
Example 3 the present invention optimizes the reaction conditions of the kit by orthogonal assay design
Orthogonal tables were selected based on 4 factors such as the coating concentration of the antigen Proteomer subbunit alpha type1 (four coating concentrations of 50. mu.g/ml, 90. mu.g/ml, 120. mu.g/ml, 150. mu.g/ml), each reaction time (15min, 30min, 45min) and temperature (25 ℃, 37 ℃), the optimal dilution of enzyme-labeled secondary antibody (four dilutions of 1:100, 1:500, 1:1000, 1: 1500), etc., each factor repeatedly determining standard positive serum and standard negative serum at 2 levels, and the ratio (P/N) of the highest light signal value (P) of positive serum to the lowest light signal value (N) of negative serum was selected. The optimal antigen package Protezome subunit alpha type1 of the kit is obtained through orthogonal design, the concentration is 90 mu g/ml, the optimal antigen-antibody reaction temperature of the anti-Protezome subunit alpha type1-IgG antibody kit for solid-phase membrane immunodetection is 25 ℃, the optimal antigen-antibody reaction time is 30min, and the optimal working dilution of the optimal labeled anti-human IgG antibody is 1:1000, parts by weight; the kit for detecting the anti-proteosome submicron alpha type1-IgG antibody by magnetic particle chemiluminescence immunoassay has the optimal antigen-antibody reaction temperature of 37 ℃, the optimal antigen-antibody reaction time of 15min and the optimal working dilution of the optimal labeled anti-human IgG antibody of 1: 1000.
example 4 preparation of solid phase Membrane immunoassay kit for detecting anti-protesome subunit alpha type1-IgG antibody
4.1 composition of solid phase membrane immunoassay kit for detecting anti-proteosome subbunit alpha type1-IgG antibody:
1. antigen: recombinant protein Proteasome subunit alpha type1
2. Solid phase carrier: sataurus CN140 nitrocellulose membrane
3. Positive quality control (standard): human anti-GST tag immunoglobulin G (purchased from Huzhou Yingchuang)
4. Negative quality control product: serum for health physical examination person
5. Labeling the antibody: biotin-labeled anti-human IgG antibody
6. Antigen diluent
7. Sample dilution buffer
8. Antibody diluent
9. Cleaning solution
10. Enzyme working solution: alkaline phosphatase-streptavidin
11. Substrate color developing solution: BCIP color developing solution.
4.2 detection procedure of the solid phase membrane immunoassay kit for detecting anti-proteosome subustit alpha type1-IgG antibody is as follows:
4.2.1 coating, sealing: placing 8 μ l of proteosome subbunit alpha type1 antigen direct contact with concentration of 90 μ g/ml on a nitrocellulose membrane, drying in a 37 ℃ incubator for 30min, placing the nitrocellulose membrane in a detection plate, adding 200 μ l of 5% BSA in a 37 ℃ incubator for sealing for 30min, discarding the sealing solution, and washing with a washing solution for 2 times;
4.2.2 antigen incubation: adding 10 μ l of antibody standard or serum to be detected diluted with diluent into the detection plate, performing negative control and positive control, incubating at 25 deg.C for 30min, and arranging 3 parallel holes for each sample;
4.2.3 Secondary antibody incubation: discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding 20 μ l of 1:1000 biotin-labeled anti-human IgG antibody, and incubating at 25 deg.C for 30 min;
4.2.4 color development: discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding 500 μ l alkaline phosphatase-streptavidin, incubating at room temperature for 20min, discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding BCIP color developing solution, reacting at room temperature for 20min, washing the detection plate with running water, and terminating the enzyme reaction. And (3) taking out the test nitrocellulose membrane strip, drying the membrane strip by using a blower, qualitatively judging by using a colorimetric card with naked eyes, and drawing a standard curve to perform semi-quantitative analysis on the antibody level of the proteosome subnitrile type1-IgG in the serum by using an analysis software carried by a developing instrument with the reference standard substance concentration as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate, wherein the positive one is shown in figure 3, or the membrane strip is placed on the developing instrument for scanning.
Example 5 preparation of magnetic particle chemiluminescence immunoassay kit for detecting anti-Proteasome outbonit alpha type1-IgG antibody
5.1 magnetic particle chemiluminescence immunoassay kit for detecting anti-protesome subbunit alpha type1-IgG antibody comprises:
1. antigen: recombinant protein Proteasome subunit alpha type1
2. Solid phase carrier: magnetic fine particles of carboxyl functional group
3. Positive quality control (standard): human anti-GST tag immunoglobulin G (purchased from Huzhou Yingchuang)
4. Negative quality control product: serum for health physical examination person
5. Labeling the antibody: acridinium ester labeled anti-human IgG antibody
6. Antigen diluent
7. Sample dilution buffer
8. Antibody diluent
9. Cleaning solution
10. Pre-excitation liquid: h2O2
11. Excitation liquid: NaOH
5.2 detection principle of magnetic particle chemiluminescence immunoassay kit for detecting anti-protesome subustit alpha type1-IgG antibody
The chemiluminescence immunoassay kit is an analysis method combining a magnetic separation technology, an immunoassay technology and a chemiluminescence technology. The kit adopts an indirect method to quantitatively analyze and detect the anti-protesome subbnit alpha type1-IgG antibody in human serum: firstly, mixing magnetic particle liquid with a diluted sample, binding a specific anti-proteosome subbnit alpha type1-IgG antibody to the magnetic particles coated by the proteosome subbnit alpha type1 antigen, washing, adding an acridinium ester labeled anti-human IgG antibody to form a compound of the magnetic particles coated by the proteosome subbnit alpha type1 antigen-the anti-proteosome subbnit alpha type1-IgG antibody-the acridinium ester labeled anti-human IgG antibody, separating the unbound substances from the compound formed by immunoreaction under the action of an external magnetic field, discarding supernatant, washing the precipitated compound, adding the compound, washing the precipitated compound, and washingPre-excitation liquid (H)2O2) Performing luminescence reaction with exciting liquid (NaOH), wherein under alkaline condition, acridine ester molecule is attacked by hydrogen peroxide to generate dioxyethane which is unstable and decomposed into CO2And an electronically excited state of N-methylacridone which emits light having a wavelength of 430nm when it returns to the ground state, and the intensity of the emitted light is collected using a chemiluminescence apparatus. The concentration of the anti-Proteasome subbnit alpha type1-IgG antibody is in direct proportion to the luminescence value, and the concentration of the anti-Proteasome subbnit alpha type1-IgG antibody in the serum to be detected is calculated through a calibration curve, which is shown in figure 4.
5.3 preparation of Proteasome subunit alpha type1 antigen coated magnetic particles
5.3.1 principle of coating magnetic particles with proteosome subunit alpha type1 antigen: based on the fact that carboxyl functional groups contained on the surface of the magnetic particles react with EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) solution to generate unstable amino active O-acylurea intermediate, the intermediate reacts with NHS (N-hydroxysuccinimide) to generate semi-stable amino active NHS ester, and the semi-stable amino active NHS ester reacts with amino on the antigen protein subunit type1 to form antigen protein subunit type alpha type1 coated magnetic particles, as shown in figure 5.
5.3.2EDC/NHS activated carboxyl magnetic particles, which comprises the following steps:
a) weighing 10mg of magnetic particles, washing the magnetic particles for 3 times by using 20mM MES, separating by using a magnet, and removing a supernatant;
b) resuspending the washed magnetic particles in 100. mu.l of 20mM MES to give a final magnetic particle concentration of 100 mg/ml;
c) sequentially adding 50 μ l of 20mg/ml EDC and 50 μ l of 24mg/ml Sμ lfo-NHS prepared by phosphate buffer solution into the cleaned magnetic particles, fully mixing, standing and activating at room temperature for 30 min;
d) after the action of the external magnetic field, the supernatant is discarded, 400 mul of 0.05M phosphate buffer solution is taken to wash the magnetic particles, and 400 mul of preservation solution is added for constant volume preservation and standby.
5.3.3 crosslinking the activated magnetic particles with the antigen protein protease subunit alpha type1, adding pre-cooled 1ml of 20mM MES into the activated magnetic particle solution, and continuously cleaning the magnetic particles for 2 times; adding 200 mu l of 2mg/ml antigen protein proteosome subBunit alpha type1 into the activated magnetic particles, fully and uniformly mixing, and standing for reaction for 16 hours at room temperature; after the reaction is finished, adding a PBS buffer solution with the pH of 7.4 and containing 0.2 percent Tween20, and repeatedly washing the magnetic particles for 2 times; then adding PBS buffer solution with pH of 7.4 containing 0.2% Tween20 and 0.2% BSA to a final concentration of 10mg/ml, mixing, standing at room temperature for 30 min; after the reaction is finished, the supernatant is discarded, and the magnetic particles are resuspended in PBS buffer with pH7.4 containing 0.2% Tween20 and 0.2% BSA, so that the activated magnetic particles are crosslinked with the antigen protein Protesome subunit alpha type 1.
5.4 preparation of acridinium ester labeled anti-human IgG antibody, which comprises the following steps:
a) preparing 2mg/mL acridinium ester solution by using dimethylformamide;
b) preparing 1mg/mL anti-human IgG antibody by using 0.2M (pH8.0) carbonate buffer solution;
c) taking acridinium ester with a molar ratio of 4:1 and an anti-human IgG antibody, fully and uniformly mixing, and reacting for 40 min;
d) the reaction was stopped by adding 20. mu.l of carbonate buffer containing 5% lysine;
e) desalting and removing impurities to obtain the acridinium ester labeled anti-human IgG antibody solution with high purity.
5.5 step of detecting anti-proteosome subbunit alpha type1-IgG antibody in serum by magnetic particle chemiluminescence immunoassay kit
5.5.1 adding the serum to be detected into 100 mul of diluted serum to be detected or IgG standard substance resisting GST label, adding 100 mul of magnetic particle solution of coating antigen protein subunit subbnit alpha type1, reacting for 15min at 37 ℃, and simultaneously making negative and positive controls;
5.5.2 adding 400 u l washing liquid to the labeled antibody and washing for 3 times x 1min, adding 100 u l acridinium ester labeled anti-human IgG antibody diluted 1:1000, reacting at 37 ℃ for 15 min;
5.5.3 Signal detection the precipitated complexes were washed after discarding the supernatant, washed 3 times with 400. mu.l of wash solution x 1min, and 100. mu.l of pre-excitation solution (H) was added2O2) And 100. mu.l of a trigger solution (NaOH) were reacted. Tong (Chinese character of 'tong')Detecting the luminescence signal by a chemiluminescence meter, and recording the luminescence value. The concentration of the antibody against the proteosome subbnit alpha type1-IgG in the serum to be detected is in direct proportion to the luminous value, and the concentration of the antibody against the proteosome subbnit alpha type1-IgG in the serum to be detected is calculated through a standard curve.
Example 6 clinical application of kit for detecting serum anti-Proteasome subbunit alpha type1-IgG antibody
6.1 Subjects included patients diagnosed with various types of nephropathy from 6 months in 2018 to 6 months in 2020, including 466 autoimmune Nephrotic Syndrome (NS), 168 Henoch-Schonlein purpura (HSP), 137 Henoch-Schonlein purpura nephritis (HSPN), 133 IgA nephropathy (IgAN), and 195 healthy children (NC) of the same age. Serum samples were taken from various renal patients and healthy controls. All subjects received a first serum sample collection prior to no immunosuppressive treatment.
6.2 detection of anti-Protezome subbnit alpha type1-IgG antibodies in various nephrotic patients the kit of the present invention was used to detect the levels of anti-Protezome subbnit alpha type1-IgG antibodies in the serum of patients diagnosed with various nephropathies from 6 months in 2018 to 6 months in 2020, including 466 cases of autoimmune nephrotic syndrome, 168 cases of allergic purpura, 137 cases of purpuric nephritis, 133 cases of IgA nephropathy and 195 healthy children at the same time, showing that the anti-Protezome subbnit alpha type1-IgG antibodies in autoimmune nephrotic syndrome patients were positive, while the anti-Protezome subbnit alpha type1-IgG antibodies in purpura nephritis, allergic purpura, IgA nephropathy patients and healthy children were negative, as shown in FIG. 6.
6.3 evaluation of value of anti-Protezome subunit alpha type1-IgG antibody as a serological marker for diagnosis of autoimmune nephrotic syndrome patients by ROC curve analysis of detection results of anti-Protezome subunit alpha type1-IgG antibody in 6.2 patients with autoimmune nephrotic syndrome by ROC curve to evaluate application value of anti-Protezome subunit alpha type1-IgG antibody in diagnosis of autoimmune nephrotic syndrome. The results show that the anti-proteosome subunit alpha type1-IgG antibody is a good serological marker for diagnosing patients with autoimmune nephrotic syndrome, and the sensitivity of the anti-proteosome subunit alpha type1-IgG antibody (using a cut-off value larger than 32.2 as a standard) as a serological marker for diagnosing patients with autoimmune nephrotic syndrome is 74.4%, the specificity is 80.8%, and the area under the curve is 0.832, which is shown in FIG. 7.
Sequence listing
<110> Zhejiang university college of medicine subsidiary children hospital
<120> kit for detecting anti-proteasome subunit alpha 1-IgG antibody
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> protein subunit subustit alpha type1 protein (Artificial sequence Unknow)
<400> 1
Met Phe Arg Asn Gln Tyr Asp Asn Asp Val Thr Val Trp Ser Pro Gln
1 5 10 15
Gly Arg Ile His Gln Ile Glu Tyr Ala Met Glu Ala Val Lys Gln Gly
20 25 30
Ser Ala Thr Val Gly Leu Lys Ser Lys Thr His Ala Val Leu Val Ala
35 40 45
Leu Lys Arg Ala Gln Ser Glu Leu Ala Ala His Gln Lys Lys Ile Leu
50 55 60
His Val Asp Asn His Ile Gly Ile Ser Ile Ala Gly Leu Thr Ala Asp
65 70 75 80
Ala Arg Leu Leu Cys Asn Phe Met Arg Gln Glu Cys Leu Asp Ser Arg
85 90 95
Phe Val Phe Asp Arg Pro Leu Pro Val Ser Arg Leu Val Ser Leu Ile
100 105 110
Gly Ser Lys Thr Gln Ile Pro Thr Gln Arg Tyr Gly Arg Arg Pro Tyr
115 120 125
Gly Val Gly Leu Leu Ile Ala Gly Tyr Asp Asp Met Gly Pro His Ile
130 135 140
Phe Gln Thr Cys Pro Ser Ala Asn Tyr Phe Asp Cys Arg Ala Met Ser
145 150 155 160
Ile Gly Ala Arg Ser Gln Ser Ala Arg Thr Tyr Leu Glu Arg His Met
165 170 175
Ser Glu Phe Met Glu Cys Asn Leu Asn Glu Leu Val Lys His Gly Leu
180 185 190
Arg Ala Leu Arg Glu Thr Leu Pro Ala Glu Gln Asp Leu Thr Thr Lys
195 200 205
Asn Val Ser Ile Gly Ile Val Gly Lys Asp Leu Glu Phe Thr Ile Tyr
210 215 220
Asp Asp Asp Asp Val Ser Pro Phe Leu Glu Gly Leu Glu Glu Arg Pro
225 230 235 240
Gln Arg Lys Ala Gln Pro Ala Gln Pro Ala Asp Glu Pro Ala Glu Lys
245 250 255
Ala Asp Glu Pro Met Glu His
260
Claims (6)
1. The application of the proteasome subunit alpha 1 antigen protein in preparing a kit for detecting the autoimmune nephrotic syndrome is characterized in that the kit consists of the proteasome subunit alpha 1 antigen protein, a solid-phase carrier, a labeled antibody, an antigen diluent, a sample diluent, an antibody diluent, a substrate color developing agent, a washing solution, a standard substance, a positive quality control product and a negative quality control product; the sequence of the antigen protein protease subunit alpha 1 is shown as SEQ ID: 1; the labeled antibody is a secondary antibody labeled by enzyme, a chemiluminescent agent or biotin; the proteasome subunit alpha 1 antigen protein is provided with a tag peptide; the standard substance and the positive quality control substance are recombinant human anti-tag peptide immunoglobulin G, or an anti-proteasome subunit alpha 1-IgG antibody is extracted from serum to be used as the positive quality control substance and the standard substance, and the negative quality control substance is serum of a healthy physical examiner; autoimmune nephrotic syndrome is diagnosed by detecting antibodies against proteasome subunit alpha 1-IgG in a serum sample from a patient.
2. The use of claim 1, wherein said tag peptide comprises: a GST tag, a c-Myc tag, a His tag, a Flag tag, or a biotin tag.
3. The use according to claim 1, wherein the proteasome subunit α 1 antigenic protein is purified by nickel column affinity chromatography, molecular sieve chromatography, ion exchange column chromatography or hydrophobic column chromatography.
4. The use of claim 1, wherein the antigen proteasome subunit α 1 is immobilized on a solid support selected from the group consisting of nitrocellulose membranes, nylon membranes, ion exchange resins, polystyrene microwell plates, and magnetic beads.
5. The use according to claim 4, characterized in that the proteasome subunit α 1 antigen protein is immobilized on the solid support in a direct manner by physical adsorption or chemical binding.
6. The use according to claim 1, wherein the substrate colour developer is TMB, AMPPD, 4-MUP, BCIP; the antigen diluent is 1x PBS pH7.4 containing 163mM NaCl and 1% TritonX-100; the sample dilution buffer was 0.01M PBS ph7.4 containing 10% BSA; the antibody diluent is 0.01M PBS pH7.4 containing 1M D-glucose, 2% glycerol, 0.35% Tween 20; the washing solution is as follows: 1 XPBS pH7.4 containing 163mM NaCl, 10% glycerol, 1% TritonX-100.
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| CN202110743551.7A CN113447659B (en) | 2021-07-01 | 2021-07-01 | Kit for detecting anti-proteasome subunit alpha 1-IgG antibody |
| CN202210532629.5A CN114895041A (en) | 2021-07-01 | 2021-07-01 | Kit for detecting anti-proteasome subunit alpha 1-IgG antibody |
| US17/856,033 US20230333115A1 (en) | 2021-07-01 | 2022-07-01 | KIT FOR DETECTING ANTI-PROTEASOME SUBUNIT ALPHA TYPE 1-IMMUNOGLOBULIN G (IgG) ANTIBODY |
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| CN202210532629.5A Division CN114895041A (en) | 2021-07-01 | 2021-07-01 | Kit for detecting anti-proteasome subunit alpha 1-IgG antibody |
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| CN114910650A (en) * | 2022-05-07 | 2022-08-16 | 浙江大学 | Application of reagent for detecting anti-moesin-IgG antibody in preparation of kit for detecting vascular endothelial injury |
| CN114924081A (en) * | 2022-05-07 | 2022-08-19 | 浙江大学 | Application of neuroblast differentiation related protein-IgG in preparation of vascular endothelial injury kit |
| CN114994330A (en) * | 2022-05-07 | 2022-09-02 | 浙江大学 | Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof |
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| CN114895041A (en) | 2022-08-12 |
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Application publication date: 20210928 Assignee: Century Yishu (Hangzhou) Medical Diagnostic Technology Co.,Ltd. Assignor: ZHEJIANG University Contract record no.: X2023980035174 Denomination of invention: A method for detecting anti proteasome subunits a 1-IgG antibody kit Granted publication date: 20220517 License type: Exclusive License Record date: 20230505 |
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